The present invention relates to Serrate genes and their encoded protein products, as well as derivatives and analogs thereof. Production of Serrate proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.
Genetic analyses in Drosophila have been extremely useful in dissecting the complexity of developmental pathways and identifying interacting loci. However, understanding the precise nature of the processes that underlie genetic interactions requires a knowledge of the protein products of the genes in question.
Embryological, genetic and molecular evidence indicates that the early steps of ectodermal differentiation in Drosophila depend on cell interactions (Doe and Goodman, 1985, Dev. Biol. 111:206-219; Technau and Campos-Ortega, 1986, Dev. Biol. 195:445-454; Vassin et al., 1985, J. Neurogenet. 2:291-308; de la Concha et al., 1988, Genetics 118:499-508; Xu et al., 1990, Genes Dev. 4:464-475; Artavanis-Tsakonas, 1988, Trends Genet. 4:95-100). Mutational analyses reveal a small group of zygotically-acting genes, the so called neurogenic loci, which affect the choice of ectodermal cells between epidermal and neural pathways (Poulson, 1937, Proc. Natl. Acad. Sci. 23:133-137; Lehmann et al., 1983, Wilhelm Roux""s Arch. Dev. Biol. 192:62-74; Jxc3xcrgens et al., 1984, Wilhelm Roux""s Arch. Dev. Biol. 193:283-295; Wieschaus et al., 1984, Wilhelm Roux""s Arch. Dev. Biol. 193:296-307; Nxc3xcsslein-Volhard et al., 1984, Wilhelm Roux""s Arch. Dev. Biol. 193:267-282). Null mutations in any one of the zygotic neurogenic locixe2x80x94Notch (N), Delta (D1), mastermind (mam), Enhancer of Split (E(spl), neuralized (neu), and big brain (bib)xe2x80x94result in hypertrophy of the nervous system at the expense of ventral and lateral epidermal structures. This effect is due to the misrouting of epidermal precursor cells into a neuronal pathway, and implies that neurogenic gene function is necessary to divert cells within the neurogenic region from a neuronal fate to an epithelial fate. Serrate has been identified as a genetic unit capable of interacting with the Notch locus (Xu et al., 1990, Genes Dev. 4:464-475). These genetic and developmental observations have led to the hypothesis that the protein products of the neurogenic loci function as components of a cellular interaction mechanism necessary for proper epidermal development (Artavanis-Tsakonas, S., 1988, Trends Genet. 4:95-100).
Mutational analyses also reveal that the action of the neurogenic genes is pleiotropic and is not limited solely to embryogenesis. For example, ommatidial, bristle and wing formation, which are known also to depend upon cell interactions, are affected by neurogenic mutations (Morgan et al., 1925, Bibliogr. Genet. 2:1-226; Welshons, 1956, Dros. Inf. Serv. 30:157-158; Preiss et al., 1988, EMBO J. :3917-3927; Shellenbarger and Mohler, 1978, Dev. Biol. 62:432-446; Technau and Campos-Ortega, 1986, Wilhelm Roux""s Dev. Biol. 195:445-454; Tomlison and Ready, 1987, Dev. Biol. 120:366-376; Cagan and Ready, 1989, Genes Dev. 3:1099-1112).
Sequence analyses (Wharton et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell. Biol. 6:3094-3108; Vxc3xa4ssin, et al., 1987, EMBO J. 6:3431-3440; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735) have shown that two of the neurogenic loci, Notch and Delta, appear to encode transmembrane proteins that span the membrane a single time. The Notch gene encodes a xcx9c300 kd protein (we use xe2x80x9cNotchxe2x80x9d 1 to denote this protein) with a large N-terminal extracellular domain that includes 36 epidermal growth factor (EGF)-like tandem repeats followed by three other cysteine-rich repeats, designated Notch/lin-12 repeats (Wharton, et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell. Biol. 6:3094-3108; Yochem, et al., 1988, Nature 335:547-550). Delta encodes a xcx9c100 kd protein (we use xe2x80x9cDeltaxe2x80x9d to denote DLZM, the protein product of the predominant zygotic and maternal transcripts; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735) that has nine EGF-like repeats within its extracellular domain (Vassin, et al., 1987, EMBO J. 6:3431-3440; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735). Molecular studies have lead to the suggestion that Notch and Delta constitute biochemically interacting elements of a cell communication mechanism involved in early developmental decisions (Fehon et al., 1990, Cell 61:523-534).
The EGF-like motif has been found in a variety of proteins, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518). In particular, this motif has been found in extracellular proteins such as the blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7:2053-2061; Furie and Furie, 1988, Cell 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al., 1988, Cell 55:1047-1059), and in some cell-surface receptor proteins, such as thrombomodulin (Suzuki et al., 1987, EMBO J. 6:1891-1897) and LDL receptor (Sudhof et al., 1985, Science 228:815-822). A protein binding site has been mapped to the EGF repeat domain in thrombomodulin and urokinase (Kurosawa et al., 1988, J. Biol. Chem 263:5993-5996; Appella et al., 1987, J. Biol. Chem. 262:4437-4440).
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present invention relates to nucleotide sequences of Serrate genes (Drosophila Serrate and related genes of other species), and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided. In a specific embodiment, the Serrate protein is a human protein.
The invention relates to Serrate derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) Serrate protein. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with Serrate for binding) to an anti-Serrate antibody], immunogenicity (ability to generate antibody which binds to Serrate), ability to bind (or compete with Serrate for binding) to Notch or other toporythmic proteins or fragments thereof (xe2x80x9cadhesivenessxe2x80x9d), ability to bind (or compete with Serrate for binding) to a receptor for Serrate. xe2x80x9cToporythmic proteinsxe2x80x9d as used herein, refers to the protein products of Notch, Delta, Serrate, Enhancer of split, and Deltex, as well as other members of this interacting gene family which may be identified, e.g., by virtue of the ability of their gene sequences to hybridize, or their homology to Delta, Serrate, or Notch, or the ability of their genes to display phenotypic interactions.
The invention further relates to fragments (and derivatives and analogs thereof) of Serrate which comprise one or more domains of the Serrate protein, including but not limited to the intracellular domain, extracellular domain, transmembrane domain, membrane-associated region, or one or more EGF-like (homologous) repeats of a Serrate protein, or any combination of the foregoing.
Antibodies to Serrate, its derivatives and analogs, are additionally provided.
Methods of production of the Serrate proteins, derivatives and analogs, e.g., by recombinant means, are also provided.
The present invention also relates to therapeutic and diagnostic methods and compositions based on Serrate proteins and nucleic acids. The invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein xe2x80x9cTherapeuticsxe2x80x9d) include: Serrate proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the Serrate proteins, analogs, or derivatives; and Serrate antisense nucleic acids. In a preferred embodiment, a Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state. In other specific embodiments, a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.
In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or Serrate function (hereinafter xe2x80x9cAntagonist Therapeuticsxe2x80x9d) are administered for therapeutic effect. In another embodiment, Therapeutics which promote Notch and/or Serrate function (hereinafter xe2x80x9cAgonist Therapeuticsxe2x80x9d) are administered for therapeutic effect.
Disorders of cell fate, in particular hyperproliferative (e.g., cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of Notch and/or Serrate protein can be diagnosed by detecting such levels, as described more fully infra.
In a preferred aspect, a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein xe2x80x9cadhesive fragmentxe2x80x9d) of Serrate which mediates binding to a Notch protein or a fragment thereof.
As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring. For example, xe2x80x9cSerratexe2x80x9d shall mean the Serrate gene, whereas xe2x80x9cSerratexe2x80x9d shall indicate the protein product of the Serrate gene.
FIGS. 1A-1F. Phenotypic interactions between Notch and Serrate. (FIG. 1A) wa spl wing blade showing characteristic wild-type symmetry, venation, and marginal wing bristles and hairs. (FIG. 1B) nd/Y male. Distal wing notches and loss of posterior hairs are evident. (FIG. 1C) SerD/+ heterozygote. Note similarity to nd/Y wing blade in (FIG. 1D) nd/Y; SerD/+ transheterozygote wing blade. Mutant wing shows typical xe2x80x9cfig leafxe2x80x9d shape, distorted wing veins, and loss of the majority of marginal bristles and hairs, with the exception of the anterodistal wing margin. (FIG. 1E) +/Y; SerD/Dp(3R)CosP479BE (N+) male. The extra N+ copy suppresses the heterozygous SerDdominant phenotype (compare to FIG. 1C). Also note suppression of the Confluens phenotype (see text). (FIG. 1F) SerD/SerD homozygote. Note the increased severity of the phenotype relative to SerD/+ (compare to FIG. C).
FIG. 2. Molecular map of the Serrate-encoding region. Approximately 85 kb of cloned genomic DNA from the 97F chromosomal region are presented along with the restriction sites of three enzymes [(B) BamHI; (E) EcoRI; (H) HindIII]. The locations of individual DNA alterations associated with Serrate allelic breakpoints are displayed above the genomic DNA (for descriptions of mutant alleles, see Section 6, infra; (rev 3 and rev 2-11) Serrev 3 and Serrev 2-11, respectively, (R128) T(Y:3)R128. The shaded box from coordinates 0 to +3 represents the region of EGF homology detectable by Southern hybridization. The BamHI site adjacent to the EGF homology was arbitrarily chosen as position 0. Map orientation is with the centromere to the left. At the bottom of the figure are shown the individual recombinant phage isolates. The C1 and C3 cDNAs together constitute the larger of the two Serrate messages (xcx9c5.6 kb). Intron positions and coding capacities have been approximated solely upon cross hybridization of the cDNAs with the genomic DNA regions.
FIGS. 3A-3F. Serrate sequence analysis. The complete 5561 bp sequence (SEQ ID NO:1) derived from cDNAs C1 and C3 is shown. Nucleotide numbering is at left, amino acid numbering of the predicted open reading frame (ORF) is at right. The deduced protein product appears to be a transmembrane protein of 1404 amino acids (SEQ ID NO:2). Hydrophobic regions are denoted inside dashed boxes; amino acids 51 to 80 represent the likely signal peptide; amino acids 542 to 564 represent the potential membrane associated region; amino acids 1221 to 1245 represent the putative transmembrane domain. The first cysteine of each of the fourteen EGF-like repeats is denoted with a solid black box, and each repeat is underlined. The partial EGF-like repeat is considered xe2x80x9cdegenerate,xe2x80x9d since the fourth cysteine residue of this repeat has been changed to lysine (shown in boldface type at amino acid position 268). The initial cysteine of this repeat is denoted with an open box (amino acid 284), and the repeat is underlined. Amino acid insertions occur in the fourth, sixth, and tenth EGF-like repeats and are denoted by hatched underlines.
FIGS. 4A-4C. The Serrate transcript and deduced protein product. (FIG. 4A) The composite transcript shown was constructed from the C1 and C3 cDNAs, which overlap by 109 bp. Selected restriction enzyme cleavage sites are shown. The hatched box represents the 4212 bp ORF. Open boxes represent the 442 bp 5-untranslated leader and 900 bp 3xe2x80x2-trailer sequence. (FIG. 4B) Kyte-Doolittle hydropathy plot of the deduced 1404 amino acid protein. (SP) Putative signal peptide; (MA) potential membrane associated region; (TM) likely transmembrane domain. (FIG. 4C) Cartoon representation of the gross structural features of the predicted Serrate protein. The darkly shaded region, including the partial EGF-like repeat (PR) is xcx9c250 amino acids in length and homologous to the Delta protein. Bracketed EGF-like repeats labeled (A, B, and C) contain insertions of amino acids and thus differ from the canonical EGF-like structure. Other features of the protein include the signal peptide (SP), a cysteine rich region, a transmembrane domain (TM), and an intracellular region of xcx9c160 amino acids.
FIG. 5. Temporal profile of Serrate transcript accumulation. Each lane contains five xcexcg of poly(A)+ RNA. The stage of the embryonic RNAs is denoted in hours after egg laying; (L1, L2, and L3) RNA from the first, second and third larval instar periods; (EP and LP) early and late pupal stages; (M and F) adult male and female RNAs, respectively. A composite cDNA subclone (constructed from C1 and C3) was used as a hybridization probe. Serrate transcription is represented primarily as a 5.5 kb and 5.6 kb doublet beginning at 4-8 hours of embryogenesis. A transient 3.4 kb transcript is observed only during 2-4 hr of embryogenesis. The pupal and adult RNAs were fractionated on a separate gel for a longer period of time for better resolution. Equivalent loadings of RNA were noted by ethidium bromide staining of the RNA gels and confirmed by subsequent probing with an actin SC probe shown at bottom; (Fyrberg et al., 1983, Cell 33:115-123). Minor bands were not consistently observed in other blots and may reflect other EGF-homologous transcripts
FIGS. 6A-6L. Whole-mount in situ Serrate transcripts. Embryos are oriented with anterior to the left and dorsal side up unless otherwise noted. (FIG. 6A) Dorsal view of an early stage embryo (mid-dorsal focal plane). Earliest expression occurs in the ectoderm of the foregut (FG) and presumptive clypeolabrum (CL). (FIG. 6B) Dorsal view of a germ band-extended embryo (late stage 10). Additional expression occurs near the proctodeum (PR), within the eighth (A8) and ninth (A9) abdominal segments, and in the labial and maxillary primordia (arrow). (FIG. 6C) Lateral view of an early stage 11 embryo. The lateral (LE) and ventral (VE) expression patterns are out of register and do not include the tracheal pits (TP). (FIG. 6D) Germ band-extended embryo (mid stage 11) dissected and flattened such that the dorsal surfaces are at the lateral edges. Extensive expression is observed between the labial (LB), maxillary (MX), and mandibular (MN) lobes, and within the hypopharynx (HP) and clypeolabrum (CL). Expression is also apparent in the salivary gland placodes (SP) that have moved to the ventral midline. Note relationship between lateral and ventral patterns and elaboration of expression in the tail region [presumptive telson (TL)]. (FIG. 6E) Germ band-retracting embryo (stage 12; lateral view). Lateral expression (LE) is beginning to coalesce. (FIG. 6F) Lateral view of a germ band-retracted embryo (stage 13). The lateral expression is beginning to extend both dorsally and ventrally in each thoracic and abdominal segment and is most pronounced in the first thoracic segment (T1). A portion of the lateral expression now appears to include the presumptive trachea (T). Ventrally, note different expression (VE) patterns in the thoracic versus abdominal segments. (FIG. 6G) Lateral view of an early stage 14 embryo. Outline of the presumptive trachea (T) is distinct from the overlying epidermal expression. Arrows denote the zigzag pattern of lateral expression. (FIG. 6H) Dissected embryo (stage 14) opened along the dorsal midline and laid flat. Two areas of hindgut expression (HG1 and HG2) are apparent; HG1 occurs near the origin of the Malpighian tubules. (FIG. 6I) Ventral view of a stage-16 embryo focusing on the ventral nerve cord (VNC). Earlier expression in the salivary gland placodes (SP in FIG. 6D) now constitutes the SD. Expression in the proventriculus (PV) and the maxillary/mandibular region (MX/MN) is slightly out of focus. (FIG. 6J) Dorsomedial focal plane of same embryo as in FIG. 6I; head involution is nearly complete. The in-pocketings of expression in the thoracic segments (T1, T2, and T3) may represent imaginal disc primordia. Pharyngeal expression (PH) is a combination of clypeolabrum and hypopharyngeal expression noted earlier. FIG. 6K Dorsal view of the same embryo as in FIG. 6I and FIG. 6J. Note individual expressing cells in the brain lobes (BC). Expression in the fully differentiated trachea (T) and hindgut (H1) is evident. (FIG. 6L) Flattened preparation of early stage 16 embryo. Expression within the telson (TL) now constitutes a ring around the presumptive anal pads.
FIG. 7. Amino acid comparison of amino-terminal Serrate-Delta homology. Conserved regions are indicated at the top of the figure (*=identical amino acids; xe2x80x2=conservative changes in sequence). Serrate (see SEQ ID NO:2) is shown above line, Delta (SEQ ID NO:4) below. The sequence begins at Serrate amino acid position 59; the partial EGF-like repeat of both Serrate and Delta is boxed. The Serrate amino acid sequence (amino acids 79-282 of FIGS. 3A-3F) placed into the chimeric xcex94EGF Notch construct and determined to be sufficient for Notch binding is presented in boldface type. The positions of the synthetic degenerate primers (designated FLE1 through FLE4R) are shown; refer to FIGS. 8A-8C for nucleotide composition.
FIGS. 8A-8C. Nucleotide comparison of amino-terminal Serrate-Delta homology. The nucleotide sequence corresponding to the amino acid sequence in FIG. 7 is shown (Serrate sequence: see SEQ ID NO:1 ; Delta sequence: SEQ ID NO:3). The DNA encoding the partial EGF-repeat is boxed. The Serrate nucleotide sequence (nucleotides 676-1287 of FIGS. 3A-3F) placed into the chimeric xcex94EGF Notch construct determined to be sufficient for Notch binding is presented in boldface type.
FIGS. 9A-9G. Nucleotide sequence (SEQ ID NO:5) and protein sequence (SEQ ID NO:6) of Human Serrate-1 (also known as Human Jagged-1 (HJ1)).
FIGS. 10A-10G. xe2x80x9cCompletexe2x80x9d nucleotide sequence (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8) of Human Serrate-2 (also known as Human Jagged-2 (HJ2) generated on the computer by combining the sequence of clones pBS15 and pBS3-2 isolated from human fetal brain CDNA libraries. There is a deletion of approximately 120 nucleotides in the region of this sequence which encodes the portion of Human Serrate-2 between the signal sequence and the beginning of the DSL domain.
FIGS. 11A-11B. Nucleotide sequence (SEQ ID NO:9) of chick Serrate (C-Serrate) cDNA.
FIGS. 12A-12B. Amino acid sequence (SEQ ID NO:10) of C-Serrate (lacking the amino-terminus of the signal sequence). The putative cleavage site following the signal sequence (marking the predicted amino-terminus of the mature protein) is marked with an arrowhead; the DSL domain is indicated by asterisks; the EGF-like repeats (ELRs) are underlined with dashed lines; the cysteine rich region between the ELRs and the transmembrane domain is marked between arrows, and the single transmembrane domain (between amino acids 1042 and 1066) is shown in bold.
FIG. 13. Alignment of the amino terminal sequences of Drosophila melanogaster Delta (SEQ ID NO:4) and Serrate (SEQ ID NO:2) with C-Serrate (SEQ ID NO:10). The region shown extends from the end of the signal sequence to the end of the DSL domain. The DSL domain is indicated. Identical amino acids in all three proteins are boxed.
FIG. 14. Diagram showing the domain structures of Drosophila Delta and Drosophila Serrate compared with C-Serrate. The second cysteine-rich region just downstream of the EGF repeats, present only in C-Serrate and Drosophila Serrate, is not shown. Hydrophobic regions are shown in black; DSL domains are checkered and EGF-like repeats are hatched.